Stanford Project 2

总结 非小细胞肺癌（NSCLC）是一种侵袭性恶性肿瘤，其中有限的治疗选择是进一步的。 受到治疗阻力的影响。免疫治疗，特别是针对PD-1/PD-L1免疫检查点的免疫治疗， 已经改变了NSCLC治疗，在两种治疗中具有持久的反应和相对最小的副作用。 转移性疾病的二线治疗，以及最近的一线治疗。尽管这些令人印象深刻的反应 存在同样令人印象深刻但定义不明确的内在和/或获得性抗性机制。交叉立体 在不同的肿瘤类型中，靶向PD-1轴的缓解率在NSCLC患者中仅为~20- 30%。治疗既往 在未经治疗的NSCLC中，抗PD-1抗体派姆单抗的总体缓解率（ORR）仅为~45%， 在患者预选为>50% IHC PD-L1肿瘤阳性的情况下，PD-L1阴性患者也表现出抗肿瘤活性， 在NSCLC中，缓解和积极寻找替代生物标志物的目标尚未实现。因此 显著的耐药机制损害了NSCLC和其他不同的PD-1靶向药物的应答 实体瘤对于生物标记物的发现和伴随的机理定义都是难以解决的。 因此，该U 54申请的项目2直接解决了内在和后天的紧迫问题。 通过分析一个非常宝贵的治疗中队列，研究NSCLC患者对PD-1靶向免疫治疗的耐药性 纵向活检。目的1：对NSCLC免疫组分进行深度单细胞RNA-seq分析 使用高效的基于微流体珠的方案进行抗PD-1治疗中活检， 发现细胞簇、T细胞活化或耗竭状态以及对免疫疗法的转录组学见解 阻力Aim 2利用我们的3D患者源性肿瘤类器官（PDO）培养物， PD-1依赖性免疫检查点和肿瘤浸润淋巴细胞的体外功能重演 临床NSCLC活检中的TIL。在这里，我们创建了NSCLC的功能性类器官培养模型， 来自纵向活检的免疫疗法抗性，测量体外抗PD-1类器官后的TIL活化 治疗并与患者反应相关联。最后，Aim 3进行前瞻性液体活检， 外显子组测序以确定功能性调节抗PD-1抗性的突变特征， 免疫检查点这些目标利用了斯坦福大学U 54工厂与卡尔文郭的协同专业知识 （项目2 PI;类器官培养，单细胞RNA-seq），罗恩利维（肿瘤免疫疗法）和石楠Wakelee 和Suki Padda（NSCLC免疫治疗试验），均与项目1临床活检密切协调，液体 来自UCSF站点的Trever Bivona和Sourav Bandyopadhyay的活检和全外显子组测序。 总体而言，我们提出了一种综合方法，用于NSCLC中对PD-1抑制的内在和获得性耐药 通过纵向治疗活检的互补单细胞、类器官和测序分析。

SUMMARY Non-small cell lung cancer (NSCLC) is an aggressive malignancy in which limited treatment options are further compromised by treatment resistance. Immunotherapy, particularly against the PD-1/PD-L1 immune checkpoint, has transformed NSCLC treatment with durable responses and comparatively minimal side effects in both second-line treatment of metastatic disease and, recently, first line therapy. Despite these impressive responses there are equally impressive but poorly defined intrinsic and/or acquired resistance mechanisms. Across solid tumor types, the response rate targeting the PD-1 axis in unselected patients is only ~20-30%. In previously untreated NSCLC, the overall response rate (ORR) to the anti-PD-1 antibody pembrolizumab is only ~45% even with patient pre-selection for >50% IHC PD-L1 tumor positivity, PD-L1 negative patients also exhibit anti-tumor response and the active search for alternative biomarkers of response in NSCLC has been unfulfilled. Thus, the significant resistance mechanisms impairing response to PD-1-targeted agents in NSCLC and other diverse solid tumors have remained intractable to both biomarker discovery and accompanying mechanistic definition. Project 2 of this U54 application thus directly addresses the pressing issue of intrinsic and acquired resistance to PD-1-targeted immunotherapy in NSCLC through analysis of an invaluable cohort of on-treatment longitudinal biopsies. Aim 1 pursues deep single-cell RNA-seq profiling of the immune component of NSCLC anti-PD-1 on-treatment biopsies using a highly efficient, microfluidic bead-based protocol allowing unsupervised discovery of cell clusters, T cell activation or exhaustion states and transcriptomic insights into immunotherapy resistance. Aim 2 exploits our 3D Patient-Derived Tumor Organoid (PDO) cultures that represent the first in vitro functional recapitulation of the PD-1-dependent immune checkpoint and tumor infiltrating lymphocytes (TILs) within clinical NSCLC biopsies. Here, we create functional organoid culture models of NSCLC immunotherapy resistance from longitudinal biopsies, measuring TIL activation upon in vitro anti-PD-1 organoid treatment and correlating against patient response. Lastly, Aim 3 performs prospective liquid biopsy and exome sequencing to determine mutational signatures of anti-PD-1 resistance that functionally regulate immune checkpoints. These Aims utilize synergistic expertise from the Stanford site of the U54 with Calvin Kuo (Project 2 PI; organoid culture, single cell RNA-seq), Ron Levy (tumor immunotherapy) and Heather Wakelee and Suki Padda (NSCLC immunotherapy trials), all in close coordination with Project 1 clinical biopsies, liquid biopsies and whole exome sequencing from Trever Bivona and Sourav Bandyopadhyay of the UCSF site. Overall, we present a comprehensive approach to intrinsic and acquired resistance to PD-1 inhibition in NSCLC via complementary single cell, organoid and sequencing analysis of longitudinal on-treatment biopsies.